BIOL2281 Pre-Lab E2: Microscopy ( 10 pts)

Transcription

1 BIOL2281 Pre-Lab E2: Microscopy ( 10 pts) Name: Lab Section: Score: 1. (3 pts) Describe the functions for the parts listed below: Ocular lenses Concentric X-Y control knobs Aperture (iris) diaphragm 2. (1 pts) Compare/contrast prokaryotes and eukaryotes in terms of size and nuclear membrane. 3. (1 pt) How do you calculate total magnification? 4. (1 pt) Which of the following terms explains why you should only have to move the fine adjustment knob very little in order to bring the image into focus when you move to a higher power objective? a. Magnification b. Parcentric c. Parfocality d. Resolving power 5. (1 pt) Why does oil immersion microscopy provide greater resolution than microscopy without oil? 6. (2 pts) Describe the materials used to clean the microscope lenses and the prepared slides, respectively. For prepared slides: For microscope lenses: 7. (1 pt) Name one biological specimen you will be observing using wet mount techniques in this lab.

2 Experiment 2: Light Microscopy Objectives: By the end of this lab, you will be able to: 1. Handle the microscope safely and know how to clean it. 2. Explain principles and terms used in light microscopy and focusing 3. Identify and know the functions of the major parts of compound microscope. 4. Examine biological specimens using wet mount techniques. 5. Understand the distinguishing cell sizes and other structural features between prokaryotic and various eukaryotic cell types. Review the structures of animal, plant and bacterial cells at Click cell biology under contents then choose cell models. Basics of brightfield microscopes 1. Parts and functions Optical components: 1. Objective lenses: Usually includes four lenses: 4X (red band), 10X (yellow band), 40X (blue band), and 100X (white band). Other numbers on the lenses include the Numerical Aperture (N.A.), the DIN number (the tube length in mm required for the lens to work properly), and the thickness in mm of the cover slip that you should use. The Working Distance (W.D.) is the space between the objective lens and specimen when the objective lens is in focus. Notice from the optical characteristics given in the table below, that the field of view decreases with increasing magnification. Depth of field is determined by the distance from the nearest object plane in focus to that of the farthest plane also simultaneously in focus. In microscopy depth of field is very short and usually measured in units of microns. As magnification increases, depth of field decreases. Magnification 4X 10X 40X 100X Numerical Aperture* W.D. (mm) Depth of field (µm) Field of View (mm) *Numerical Aperture = nsinμ. n = Refractive index of medium between specimen and the front lens of the objective. Refractive index of air is 1.00+; the refractive index of immersion oil is 1.515) μ = 1/2 the angle of cone of light "captured" by the objective lens. 2. Ocular lenses: The lenses you look into (10X are used in this lab). 3. Substage condenser: Its function is to gather the light coming from the light source and to concentrate that light in a collection of parallel beams onto the specimen. 1

3 Mechanical/Electrical Components 1. Base: It houses the electrical components for operating and controlling the intensity of the lamp. A colorful translucent stop plate may be placed over the lamp for greater contrast. 2. Stage: The specimen is placed on top of the stage and held in place by a specimen holder. Attached to the stage are concentric X-Y control knobs which move the specimen forward/back or left/right. 3. Coarse and Fine focusing Knobs: On the lower right and left side of the stand are the concentric coarse and fine focusing Knobs. These raise or lower the stage in larger or smaller increments to bring the specimen into focus. 4. Observation tubes: either binocular or trinocular, are attached to the stand above the nosepiece. The binocular is used for viewing and the trinocular is used for viewing and /or photography. 5. Nosepiece: It holds the objective lenses of varying magnifications. The rotation of the nosepiece can bring any one of the attached objective lens into the light path. 6. Condenser Holder: It is under the stage to hold a condenser. Adjacent to the condenser holder is the knob for raising or lowering the condenser. The adjustable aperture iris diaphragm sometimes is incorporated in the condenser or just below it. It is t important to use aperture (iris) diaphragm properly in securing excellent illumination and contrast. Opening the iris diaphragm too much results in glare and loss of contrast, while closing it too far results in increased diffraction and loss of resolution. 7. Interpupillary distance adjustment: This design sets the interpupillary distance for your eyes. 8. Diopter adjustment: It is an adjustable viewfinder, on each ocular lens, to suit the eyesight of the user. 9. Phase contrast revolving turret: It is a rotating disk under the stage to use for phasecontrast optics (normal setting is at zero. When in use, the turret setting must correspond to the magnifying power of the objective being used). Phase contrast is very useful in showing details in certain specimens of unstained cells. 2. Concepts Magnification: How much larger the object appears compared to its real size. Total magnification of the specimen is determined by multiplying the magnification of the objective lens by the magnification of the ocular lens. Light microscopes can magnify up to 1000 or 1500 times. Resolving power: A measure of the clarity with which an objective can clearly separate points or lines lying close together in the specimen. The shorter the distance between points or lines, the greater the resolving power. Also, the higher the NA of the objective, the greater its resolving power is. Limited by the wavelengths of visible light, light microscopes can never resolve detail finer than ~ 0.2 microns. Contrast: A measure of the gradation in luminance that provides gray scale (or color) information. Contrast is expressed as the ratio (difference in luminance)/ (average luminance) in adjoining areas of the scene. Contrast should not to be confused with resolution. Contrast can be 2

4 enhanced by specimen staining, adjusting condenser aperture diaphragm settings and using several contrast-enhancement techniques, including differential interference contrast (DIC), darkfield, and phase contrast. 3. Operation and Handling of a Light Microscope 1) Handling and cleaning Tighten the screw between nosepiece and observing tubes before moving a microscope. Carry it with both hands one on the arm and the other supporting the base. It is always a good idea to clean all of the lenses with lens paper and lens cleaner before you begin. It is IMPERATIVE that you clean all lenses after you finish using the microscope. DO NOT use Kimwipes on the lenses. Remove all the slides and patch stops and rotate the lowest power objective in the light path when you finish. Place the microscope back to the cabinet with the arms toward you. 2) Focusing a Compound Light Microscope 1. Turn on the lamp and adjust the intensity. 2. To set the interpupillary distance for your eyes, grasp the binocular observation tubes with both hands and bring the tubes closer together (or further apart) to fuse the two circles of light into one circle. 3. With the 10X objective on the nosepiece in the light path, place a specimen slide on the stage. Raise the stage to the highest position (the 10X objective should not touch the slide under normal condition). Using your RIGHT eye only, slowly lower the stage by use of the coarse adjustment knob. Bring the image into focus and then use the fine adjustment knob to perfect the focus. Now using your left eye only, rotate the diopter adjustment ring on the left eyepiece tube without touching the focusing knobs to bring the image into focus for your left eye. 4. You should only move the fine adjustment knob very little to bring the image into focus when you move to a higher power objective because the microscope has a built-in feature which is known as parfocality. The specimen should remain at the center of the viewing field when objective lenses are rotated because this microscope is parcentric. 3) Using an Oil immersion objective (optional, with TA assistance ONLY) The 100X objective lens is used for oil immersion in order to achieve the greatest resolution. (The refractive index of oil is close to that of glass, reducing the refraction of light.) Proper technique must be followed when using oil immersion. Lenses are expensive and dried oil renders them dysfunctional! In order to use oil immersion, follow these steps: 1. Focus the specimen with the 10X objective. Then switch to the 40X objective lens and center a desired feature in the field of view. 2. Rotate 40 X objective lens away from the specimen and before rotating the 100X lens into position, (do not lower the stage) gently place a drop of immersion oil on top of the cover slip. 3. Rotate the 100X objective into the light path. 4. Using the fine adjustment only, adjust the stage until the specimen comes into focus. 3

5 5. When finished with observations with the oil immersion objective, lower the stage and rotate objective out of the light path so that you do not inadvertently dip the 40X objective into oil. 6. You must thoroughly clean the lens with lens cleaner and lens paper after using oil! Clean the prepared slides with lens cleaner and KimWipes. Do not use KimWipes on the lenses as it will scratch them. 4) Preparing a Wet mount and Staining 1. Place a drop of water on a microscope slide. 2. Use a toothpick to scrape the inside of your cheek. Roll the scraping into a drop of water on the slide. 3. Place the edge of a coverslip at an edge of the drop at a 45º angle; gently lower the coverslip into place with support of a dissecting needle to prevent trapping air bubbles. 4. In order to stain a wet mount, place a drop of methylene blue next to the coverslip and position a small piece of paper towel on its opposite edge. Diffusion will draw an appropriate amount of stain into the specimen. Be very careful when using stains. They will stain your skin, clothing, and floor as well. 5. Do not use 100X objective for wet mount. Adjust the condenser iris diaphragm to increase brightness if necessary. * This technique also applies to viewing onion skin cells Biological Specimen for Observation in today s lab: Bacterial Cells Prepared slides (prepared demo slide) Eukaryotic Cells Fresh budding yeast cells (demo slide) Mold (prepared slide) Materials per student: Amoeba (prepared slides) Diatoms (prepared slide) Spirogyra (prepared slide) Fresh onion skin (methylene blue stain) Human cheek cells (methylene blue stain) Paramecium (prepared demo slide) Volvox (prepared slide) Fresh plant stem and leaf Fresh Elodea Human Blood smear (prepared slide) one Microscope (Pick up microscope according to your sign-in number) one book of lens paper, one bottle of Lens cleaner, one box of Kim wipes A box of prepared slides, legend page for microscope, identification guide for fungi References: Mortimer Abramowitz, Microscope basics and beyond Published by Olympus America Inc., 2003, Molecular Expressions 4

6 The report of Experiment 2 should include a title page (with your name, course number, section number and the title of the experiment) and pages 5 to 9. Failure to include a title page will result in the loss of 1 point from your score. Section I: Plant Cells 1 Make a wet mount of a whole live Elodea (or Anacharis) leaf. The chloroplasts may seem to be crawling along the inner wall. This phenomenon is described in biology as cytoplasmic streaming. The purpose of the motion is to transports nutrients, proteins, and organelles within cells. Draw what you see under the 40X objective lens. Label the chloroplasts within a cell. (2 pts) Elodea leaf cells at total magnification of X 2. Make a wet mount of celery stalk. Slice a very thin cross section and place it in a drop of water on a microscope slide. Draw what you see under the 10X or 40X objective lens. Label the vascular bundles. (2 pts) Celery stalk at total magnification of X 3. Make a wet mount of onion skin and stain with methylene blue. Snap a piece of onion backward and remove of the thin piece of epidermis formed at the breaking point. Place the epidermal tissue in a drop of water on a microscope slide. Add a coverslip then stain the cells following procedures on page 4. Draw what you see under the 40X objective lens. (1 pt) Onion skin cells at total magnification of X 5

7 Section II: Animal Cells 3. Make a wet mount of your cheek cells and stain with methylene blue. Draw what you see under the 40X objective lens. Label the nucleus within a cell. (2 pts) Dispose used slides into broken glass boxes under front sinks. Human cheek (epidermis) cells at total magnification of X What causes the structural differences observed in the Elodea cells compared to the cheek cells? 4. Examine a prepared human peripheral blood smear slide. This smear was prepared with polychromatic Wright's stain, which stains both basophilic and acidophilic cellular components. Identify red blood cells (erythrocytes), the most numerous cells present. White blood cells (leukocytes) have several distinct cell types: neutrophils, eosinophils, basophils, monocytes and lymphocytes. The first three cell types are grouped together as granulocytes or polymorphonuclear (PMN) leukocytes. The latter two types are categorized as mononuclear leukocytes. Using the 10X objective, identify white blood cells (leukocytes) with lobulated nuclei, the granulocytes. The most abundant granulocytes are neutrophils. Examine with the 40X objective the nuclei and the lightly stained cytoplasmic granules of several neutrophils (Examples at WebPath). Sketch at least one leukocyte (label it/them) and some surrounding erythrocytes under the 40X objective lens. (1 pt) In the center of the field is a neutrophil. An eosinophil with a bilobed nucleus and numerous reddish granules in the cytoplasm is seen on the top-right. There is a basophil with numerous dark blue granules in the cytoplasm on the right. 6

8 Section III: Bacterial Cells 5. Prokaryotes are now divided into two Domains, the Archaea and the Bacteria. At the demonstration microscope, observe one of the three most common shapes of Bacteria: bacillus (rod), coccus (spheres), or helices under oil immersion and draw what you see. Identify the type of bacteria using the poster in lab. (1 pt) shape/type at total magnification X Visit Link: to view an animation to compare the relative sizes of cells and organisms. Section IV: Fungal Cells 6. Fungi are eukaryotic, heterotrophic, nonphotosynthetic organisms in a separate kingdom called Fungi. The majority consists of filaments called hyphae, and the network of filaments is the mycelium. Rhizopus nigricans is the common black mold that develops on stale bread and it makes sporangiospores asexually. Penicillium species have the ability to produce conidiospores as their asexual spores. Examine Rhizopus and Penicillium (the slide of mold types) using the 10X objective lens. Notice how they make different types of spores. Label the following diagrams as either Rhizopus or Penicillium (2 pts). a) b) c) Yeasts (Saccharomyces spp) have long been used as an important model organism for molecular biologists, and will continue to yield critical information on research ranging from development to aging and everything in between. Observe the wet mount slide of haploid yeast cells cultured on YED agar medium. Draw and label budding and non-budding cells. (2 pts) Yeast cells at total magnification X 7

9 Section V: Protozoan and Algal Cells 7. Kingdom Protista can be subdivided into Subkingdom Protozoa (animal-like protists) and Subkingdom Algae (plant-like protists). Algae are either unicellular, colonial, or have simple multicellular bodies called thalli, which are commonly composed of branched filaments or sheets. In this lab, you will observe three types of algae: Diatoms, Volvox and Spirogyra. Be careful with this group of slides, many slides were smashed by objective the lens in previous semesters. a) Diatoms are unicellular and unique in that they have hard cell walls of pectin, cellulose, or silicon oxide that are constructed in two halves. The two halves fit together like lid and box. The diversity and beauty of these single-celled organisms is astounding. Draw several different diatom cells under 10 or 40X objective (1 pt). Objective lens used: X b) Examine the prepared slides for Volvox and Spirogyra using the 10X objective lens. Volvox is a colonial photosynthetic algae composed of numerous small cells and daughter colonies. Note the unusual feature of the spiraling chloroplasts in Spirogyra. Draw below (2 pts): Spirogyra (under 10X objective lens) Volvox (under 10X objective lens) Are these organisms capable of photosynthesis? 7. Members of subkingdom protozoan are categorized based on their modes of locomotion. Amoebas move using pseudopodia (cellular extensions). Flagellates move using flagella (e.g. Trypanosoma spp). Ciliates move using cilia (e.g. Paramecium spp.) Others that lack motility (e.g. Plasmodium spp.) View the prepared slide of Amoeba (from your slide box) and a prepared slide of Paramecium (at the demo scope) and observe under the 10X or 40X objective lens. Draw what you see. (2 pts) Amoeba (under X objective) Paramecium (under X objective) 8

10 Label the parts of a microscope using a legend guide (provided in lab, 2 pts): 9

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12 Clean up: Remove slide from the stage. Lower the stage and rotate 4X objective to the center. Collect and examine all prepared slides upon completion of this lab. Report to your instructor any damage or concern. Have the TA check your microscope before you return your microscope to the cabinet. Clean your bench area, return materials and supply items and sign out. 11

13 Experiment 2 Microscopy BIOL2281 Parts of the Microscope Basic Microscope Techniques Observing Cells and Organisms Types of Microscopes Light Microscopes (LMs) 1. Stereomicroscope (3D): dual objective lenses Dissecting microscopes 2. Ocular lens : Monocular, binocular, trinocular heads Objective lenses:,,, Electron Microscopes (EMs) 1. Transmission: TEM 2. Scanning: SEM Objective Lens Characteristics Magnification 4X 10X 40X 100X Numerical Aperture Parts of Microscope Ocular lens Objective lens- binocular Condenser lens Concentric X-Y control knobs Aperture Iris diaphragm Light Source and adjustment Coarse adjustment knob Fine adjustment knob Interpupillary distance adjustment Diopter adjustment Parts of Microscope (cont.) Concentric X-Y control knobs - control the Condenser Lens (adjustment knob) focuses Aperture (Iris) diaphragm - controls the Interpupillary distance adjustment - adjusts the Diopter adjustment- used to suit the eyesight of the user Dr. Wenju Lin and Dr. Elizabeth Pickett Spring

14 Microscopy Terminology Magnification How much larger the object appears compared to its real size. LMs can magnify up to ~ 1000 times. Resolving power A measure of the clarity of the image; the minimum distance two points can be separated and still be distinguished. LMs can never resolve detail finer than ~ 0.2 microns Contrast Expressed as the ratio (difference in luminance)/ (average luminance) in adjoining areas of the scene. Contrast should not to be confused with resolution. Techniques with LMs Wet mounts Staining- Methylene blue Oil immersion Brightfield Darkfield Rhienberg Fluorescence Phase-contrast Wet mount technique Use a toothpick to scrape the inside of your cheek. Roll the scraping into a drop of water on a slide. To prevent trapping air bubbles, slowly lower a coverslip onto the slide with the support of a dissecting needle. Place a drop of methelene blue next to the coverslip. Position a small piece of paper towel on its opposite edge. Diffusion will draw an appropriate amount of stain into the specimen. This technique also applies to viewing onion skin cells- thin epidermis Oil Immersion Oil immersion provides greater. The refractive index of oil is close to that of glass, preventing the refraction of light. 1 drop of oil is placed on top of the coverslip. The objective lens is brought into position and creates a bridge of oil between the slide and lens. You MUST clean the lens with lens cleaner and lens paper after using oil!! Use lens cleaner and KimWipe to clean the slide. Dr. Wenju Lin and Dr. Elizabeth Pickett Spring

15 Oil Immersion (cont.) Start from lower power. Move to 40x objective lens after you achieve the best resolution. Before rotating the 100x lens into position, apply 1 drop of oil on top of the coverslip Rotate 100x lens into position. After viewing the specimen, remove the slide before you rotate 40x back to position. Side by side comparisons of dry and oil immersion : Performed by inserting an opaque disk (called a "stop") in the light path. The illumination is oblique and enters the microscope objective ONLY if refracted by the object, the background remains dark. : If the central stop is made of a colored translucent material, the image background becomes the color of that material. An endothelial cell from the bovine pulmonary artery has been labeled with three different fluorescent stains. Nucleus: blue Microtubules of cytoskeleton: green Mitochondria: red A Review of Biology Terms Prokaryotic: cells do not contain membrane-bound organelles; chromosome is not contained within a nuclear membrane. Eukaryotic: cells contain membrane-bound organelles; chromosomes are contained with a nuclear membrane. Unicellular: each cell is an individual organism. May exist as single cells or in colonies. Multicellular: organism is composed of many, differentiated cells. Autotrophic: organism is capable of producing food by photosynthesis or chemosynthesis. Heterotrophic: organism must ingest or consume other organisms for food. Whittaker s Five Kingdom Scheme Link: Click cell biology under contents then choose cell models Dr. Wenju Lin and Dr. Elizabeth Pickett Spring

16 Eukaryotic Kingdoms Bacterial Shapes (1): Cocci Bacterial Shapes (2): Bacilli Bacterial Shapes (3): Helical and Spiral Animalia: Multicellular; no cell walls; chemoheterotrophic Plantae: Multicellular; cellulose cell walls; usually photoautotrophic Fungi: Chemoheterotrophic; unicellular or multicellular; cell walls of chitin; develop from spores or hyphal fragments Protista: A catchall for eukaryotic organisms that do not fit other kingdoms Wet Mount of Cheek Cell Human Blood cells eosinophil basophil erythrocytes neutrophils monocytes lymphocytes Plant Tissues: Stems Plant Tissues: Leaves Celery Stalk Dr. Wenju Lin and Dr. Elizabeth Pickett Spring

17 Kingdom Fungi Figure 12.1 Kingdom Protista Subkingdom Protozoan Amoeba-pseudopodium Paramecium Subkingdom Algae (photosynthetic, but no tissue differentiation) Euglena Volvox Spirogyra diatom Amoeba Paramecium Diatoms In lab this week You will observe: Prepared slides Bacterial cells Human blood cells Protist cells Plant cells Fungal cells Fresh-made sample slides (wet mounts) Animal cells (from your cheek) Plant cells (Elodea, celery and onion skin) Notes and reminders: Pre-lab E2 is due when you sign in for Lab E2 Bring sharpened pencils Pages 5-10 of E2 procedure (report pages) should be printed single-sided Optional (but recommended): Bring E4 procedure and report pages to work on if you have extra time Dr. Wenju Lin and Dr. Elizabeth Pickett Spring